Abstract
Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. Here we investigate the role of the intracellular growth operon (igr), which has previously been identified as having a cholesterol-sensitive phenotype in vitro and which is important for intracellular growth of the mycobacteria. We have employed isotopically labeled low density lipoproteins containing either [1,7,15,22,26-(14)C]cholesterol or [1,7,15,22,26-(13)C]cholesterol and high resolution LC/MS as tools to profile the cholesterol-derived metabolome of an igr operon-disrupted mutant (Δigr) of M. tuberculosis. A partially metabolized cholesterol species accumulated in the Δigr knock-out strain that was absent in the complemented and parental wild-type strains. Structural elucidation by multidimensional 1H and 13C NMR spectroscopy revealed the accumulated metabolite to be methyl 1β-(2'-propanoate)-3aα-H-4α-(3'-propanoic acid)-7aβ-methylhexahydro-5-indanone. Heterologously expressed and purified FadE28-FadE29, an acyl-CoA dehydrogenase encoded by the igr operon, catalyzes the dehydrogenation of 2'-propanoyl-CoA ester side chains in substrates with structures analogous to the characterized metabolite. Based on the structure of the isolated metabolite, enzyme activity, and bioinformatic annotations, we assign the primary function of the igr operon to be degradation of the 2'-propanoate side chain. Therefore, the igr operon is necessary to completely metabolize the side chain of cholesterol metabolites.
Highlights
Cholesterol metabolism is critical in the chronic phase of Mycobacterium tuberculosis infection
M. tuberculosis resides in host granulomas where cholesterol is abundant, and it has been demonstrated that M. tuberculosis is able to metabolize cholesterol
We investigated the functional role of the igr operon through 13Cmetabolite profiling and biochemical assay of FadE28FadE29, enzymes encoded in the operon
Summary
Cholesterol metabolism is critical in the chronic phase of Mycobacterium tuberculosis infection. Conclusion: The igr operon encodes the enzymes that catalyze the final three steps in cholesterol side-chain degradation. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, imports and metabolizes host cholesterol during infection. This ability is important in the chronic phase of infection. The genes and encoded enzymes necessary for the metabolism of the A and B rings of cholesterol have been identified and characterized to varying extents [10, 13,14,15,16,17,18,19] (Fig. 1A). The intracellular growth (igr) operon is located in the 83-gene cholesterol degradation locus and is required for in vitro growth on cholesterol as a sole carbon source but is not
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